Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp and its implication for the mechanism of trimeric PNPs

The three-dimensional structure of the trimeric purine nucleoside phosphory lase (PNP) from Cellulomonas sp. has been determined by X-ray crystallograp hy. The binary complex of the enzyme with orthophosphate was crystallized i n the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 64.1 Angstrom, b = 108.9 Angstrom, c = 119.3 Angstrom and an enzymatically active trimer in the asymmetric unit. X-ray data were collected at 4 degree s C using synchrotron radiation (EMBL/DESY, Hamburg). The structure was sol ved by molecular replacement, with the calf spleen PNP structure as a model , and refined at 2.2 Angstrom resolution. The ternary "dead-end" complex of the enzyme with orthophosphate and 8-iodoguanine was obtained by soaking c rystals of the binary orthophosphate complex with the very weak substrate 8 -iodoguanosine. Data were collected at 100 K with CuKalpha radiation, and t he three-dimensional structure refined at 2.4 Angstrom resolution. Although the sequence of the Cellulomonas PNP shares only 33% identity with the calf spleen enzyme, and almost no identity with the hexameric Escheric hia coli PNP, all three enzymes have many common structural features, viz. the nine-stranded central beta-sheet, the positions of the active centres, and the geometrical arrangement of the ligands in the active centres. Some similarities of the surrounding helices also prevail. In Cellulomonas PNP, each of the three active centres per trimer is occupie d by orthophosphate, and by orthophosphate and base, respectively, and smal l structural differences between monomers A, B and C are observed. This sup ports cooperativity between subunits (non-identity of binding sites) rather than existence of more than one binding site per monomer, as previously su ggested for binding of phosphate by mammalian PNPs. The phosphate binding site is located between two conserved beta- and gamma -turns and consists of Ser46, Arg103, His105, Gly135 and Ser223, and one or two water molecules. The guanine base is recognized by a zig-zag pattern o f possible hydrogen bonds, as follows: guanine N-1 ... Glu204 O-epsilon 1.. . guanine NH2... Glu204 O-epsilon 2. The exocyclic O6 of the base is bridge d via a water molecule to Asn246 NO, which accounts for the inhibitory, but lack of substrate, activity of adenosine. An alternative molecular mechanism for catalysis by trimeric PNPs is propos ed, in which the key catalytic role is played by Glu204 (Glu201 in the calf and human enzymes), while Asn246 (Asn243 in the mammalian enzymes) support s binding of 6-oxopurines rather than catalysis. This mechanism, in contras t to that previously suggested, is consistent with the excellent substrate properties of N-7 substituted nucleosides, the specificity of trimeric PNPs versus 6-oxopurine nucleosides and the reported kinetic properties of Glu2 01/Ala and Asn243/Ala Feint variants of human PNP. (C) 1999 Academic Press.